Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris

Wysocka‐Kapcinska, Monika; Campos-Sandoval, José A.; Pal, Akos; Findlay, John B. C.

doi:10.1016/j.pep.2010.01.015

Cited by 12 publications

(13 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To test whether STRA6-GFP could bind its ligand, RBP, co-localization analysis was performed using confocal microscopy. RBP was expressed in Pichia pastoris , purified as described previously [ 29 ] and labelled with DyLight 594 for the microscopy studies (see also S1 Fig ). DyLight 594-conjugated holo-RBP was found associated only with the surface of cells that were transformed with STRA6-GFP indicating that the recombinant fusion protein is functional in situ ( Fig 1B ).…”

Section: Resultsmentioning

confidence: 99%

Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

Breen

Martin

et al. 2015

PLoS ONE

View full text Add to dashboard Cite

STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6-GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.

show abstract

Section: Resultsmentioning

confidence: 99%

Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

Breen

Martin

et al. 2015

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Retinol binding protein preparation and ROH binding ‐Recombinant RBP was produced using a Pichia pastoris yeast strain stably transfected with a pPICZ α plasmid containing the DNA sequence encoding RBP . Purified RBP was incubated at an equimolar concentration to ROH (3.45 µ) for 10 mins at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

“…Purified RBP was incubated at an equimolar concentration to ROH (3.45 µ) for 10 mins at 37 °C. ROH binding was assessed by fluorescence quenching using a Cary Eclipse fluorescence spectrophotometer (Varian) as described previously .…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The effect of retinol binding protein on the proteome of C2C12 muscle cells

Young

Leonard

Martin

et al. 2015

Diabetes Metabolism Res

View full text Add to dashboard Cite

show abstract

“…Furthermore, unlike the E. coli expression system, P. pastoris can produce foreign proteins intracellulary or extracellulary, depending on the signal sequence used in the system (70). The ability to be cultured at a high cell density, as well as the folding, processing of proteins, reduced heterologous proteins degradation and a much easier heterologous protein purification procedure compared to other bacterial expression systems, render P. pastoris more attractive as an excellent alternative to the E. coli expression system (71). Moreover, heterologous proteins expressed by the P. pastoris strain were able to be post-translationally modified as observed in mammalian expression systems without the use of expensive culture media and cell culture equipment, which always significantly raise the cost of the mammalian cell expression systems (67,68,72).…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Potential combinatorial effects of recombinant atypical chemokine receptors in breast cancer cell invasion: A research perspective

2013

View full text Add to dashboard Cite

Abstract. Apart from their major function in the coordination of leukocyte recruitment, chemokines, in cooperation with their receptors, have been implicated in the progression of various diseases including different types of cancer, affecting survival, proliferation and metastasis. A complex network of chemokines and receptors exists in the tumor microenvironment and affects tumor development in various ways where chemokines activate typical signalling pathways by binding to the respective receptors. The identification and characterization of a group of atypical chemokine receptors [D6, Duffy antigen receptor for chemokines (DARC), ChemoCentryx chemokine receptor (CCX-CKR) and CXCR7] which appear to use unique biochemical properties to regulate the biological activities of these chemokines, is useful in the effort to therapeutically manipulate chemokines in a broad spectrum of diseases in which these chemokines play a critical role. The aim of this review was to investigate the combinatorial effect of two reported atypical chemokine receptors, D6 and DARC, on breast cancer cell invasion to understand their role and therapeutic potential in cancer treatment. In this regard, findings of the present review should be confirmed via the construction of recombinant D6 and DARC clones as well as the expression of the respective recombinant proteins using the Pichia pastoris (P. pastoris) expression system is to be performed in a future study in order to support findings of the current review.

show abstract

Expression and characterization of recombinant human retinol-binding protein in Pichia pastoris

Cited by 12 publications

References 17 publications

Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

The effect of retinol binding protein on the proteome of C2C12 muscle cells

Potential combinatorial effects of recombinant atypical chemokine receptors in breast cancer cell invasion: A research perspective

Contact Info

Product

Resources

About